High-performance liquid chromatography (HPLC) is commonly used for analytical and preparative separations of biopolymers and other organic molecules. For instance, the individual components within a complex organic reaction mixture may be separated by HPLC. HPLC is performed in a pressure-resistant tube containing a stationary adsorbent which is the packing material. A pressure mechanism exerts pressure on a mobile phase applied to one end of the column and moves it through the column causing it to exit the opposite end of the column. A sample containing a mixture of compounds is injected onto the column through a sample injection port. As the sample moves through the packing material, the various components of the sample adsorb to the packing material with different affinities. The components, therefore, can elute from the column separately under appropriate conditions. On a reverse phase HPLC column the compounds within a sample are separated based on hydrophobicity.
HPLC analysis may be performed in isocratic or gradient mode. An isocratic HPLC separation is one which is carried out under a constant eluant composition. A gradient HPLC separation is characterized by a gradual change in the percentage of solvent applied to the column over time. The change in solvent often is controlled by a mixing device which mixes solvent A and solvent B to produce the HPLC solvent just prior to its movement through the column. The amount of time over which the gradient is changed from one extreme to the opposite extreme is the gradient time.
Generally in gradient chromatography it is believed that increasing the flow rate and/or decreasing the gradient time results in a loss of resolution, that is the ability of the column to separate the components within the mixture into discrete eluant fractions.
Rapid methods for the preparation and isolation of potential drug candidates using automated synthetic organic chemistry techniques to create combinatorial libraries represents an important advance in drug discovery. Certain combinatorial libraries encompass a series of compounds having common structural features but which differ in the number or type of group attached to the main structure. Each compound within a combinatorial library created by parallel synthesis is a separate sample housed in a tube or well of a microtitre plate. Once the library is completed each sample is subjected to quality control analysis to confirm that the particular sample includes the desired library component at the requisite purity. Generally this is accomplished by subjecting the samples to HPLC with UV or mass spectrometry detection, IR, NMR, or any other appropriate analytical techniques. The qualitative analysis of such combinatorial libraries by conventional HPLC requires on the average 5 to 20 minutes in order to separate various compounds within the sample.
A problem encountered with prior art methods for separation of compounds in combinatorial libraries using HPLC is the length of time required for separation of each sample. Each sample of a combinatorial library produced by parallel synthesis must be analyzed separately to determine if that sample houses the appropriate compound and/or to separate the compounds in the mixture. Each library includes thousands of samples each of which require an average run time of 10 minutes. The amount of time required to perform separations on these samples may run on the order of months using standard equipment and methodology.